1. Field of the Invention
This invention is directed to colorimetric biosensors.
2. Description of Related Art
There has been a well-recognized need for fast, reliable and inexpensive analytical techniques that would be capable of detecting the presence of chemical and/or biological analytes. Although conventional analytical techniques, such as chromatography, mass spectrometry, titration, etc. can provide reliable results, these techniques are generally time consuming, expensive, performed in a laboratory at a fixed location, and are not easily portable or adaptable for portable use.
Various chemical and/or biological sensors have been developed to overcome some of the disadvantages of conventional instrumental methods of analyte detection. These are generally composed of two distinct functional components: a sensing element and a transducer. The sensing element chemically interacts with the analyte of interest to induce changes in some detectable physicochemical property, while the transducer detects theses changes and converts them into a measurable output signal.
Most chemical and/or biological sensors can be categorized into four main categories: optical, resistive, electrochemical or acoustic mass sensing devices. Among these, preferred are optical devices that generate colorimetric, fluorimetric or fluorescence depolarization signals, wherein the molecular recognition event triggers a detectable or noticeable color change that is observable by the naked eye and/or is quantifiable by optical absorption using spectroscopic instrumentation. A particularly promising step in this direction is a recently disclosed system of conjugated polymer vesicles that are bonded together with a polydiacetylene (PDA) backbone. When conjugated to biologically interactive carbohydrates such as sialic acid and ganglioside GM1, the resulting highly colored polymerized vesicles exhibit visible binding-induced color changes in the presence of influenza virus and cholera toxin, respectively. Such color changes result from perturbation of PDA chain conformation and the extent of uninterrupted conjugation, which are typically caused by heat, interactions with reagents (including analytes), changes in pH, or mechanical stress.
Diacetylenes are rather unusual among various classes of well-known polymerizable organic functional groups in that a highly ordered state is required for their polymerization to occur. In practice their polymerization has been achieved in solid state crystal lattices, Langmuir-Blodgett (LB) films, self-assembled monolayers (SAM) or vesicles. This, usually referred to as “topochemical polymerization,” is typically initiated by heat or irradiation from an ultraviolet or gamma radiation source and proceeds by repeated 1,4-addition of diacetylene monomers in a geometrically constrained polymerization medium, as illustrated in FIG. 1. The resulting polymers have highly conjugated segments of alternating double and triple bonds along the backbone, and are usually highly colored. For example, in the case of polymerized vesicles, the predominant colors are blue, red or violet.
However, the reported PDA-vesicle system has the following limitations. First, since vesicle formation is based on self-assembly of a large number of small molecules, molecular architecture cannot be controlled directly, resulting in a variety of different shapes and sizes when lipids randomly self-assemble into vesicles. Secondly, it is reportedly difficult to immobilize lipid assemblies to surfaces. Third, lipid assemblies are unstable under certain conditions such as elevated temperatures or in the presence of organic solvents.
Significant improvements in the PDA-vesicle system were recently disclosed (Charych et. al. U.S. Pat. No. 6,022,748). By this approach, lipid assemblies were immobilized in sol-gel glasses to create interpenetrating networks of polydiacetylene and alkoxysilane/silicon dioxide domains. The resulting organization helped address some of the problems related to lipid immobilization and instability, but also showed some of its own problems. Primarily, it depends on the formation of a compatible mixture between the lipid-based sensor and a sol-gel precursor, which may not be possible for every type of sensing group formulation. Secondly, incorporation of sol-gel materials adds a significant, new step of synthetic complexity to the practical realization of this invention.
Others have reported the synthesis of crosslinkable dendritic materials containing diacetylene functional groups. For example, in Colloids and Surfaces A: Physicochemical and Engineering Aspects, Volume 171, Pages 185–197, (2000), 10,12-pentacosadiynoic acid was attached to the end-groups of generation 2 polyamidoamine (PAMAM) dendrimers, and the resulting amphiphilic multi-arm star polymers were cross-linked as monolayers on the air-water interface or in dilute colloidal suspension. The authors of this study clearly recognized that their system almost certainly did not polymerize intramolecularly, that is, within the confines of a single macromolecule. Further, although they noted previous utilization of polydiacetylenes in sensing applications, they did not indicate that their own work had any potential for chemical or biological sensors. Similarly, in Polymer Preprints, Volume 43, Number 2, Page 450 (2002), octadeca-5,7-diyn-1-ol was attached to several small polyamide dendrons and polymerized to insoluble network materials. As in the previous example, the potential sensing applications of these self-assembled dendrons were not discussed. Moreover, when the —NH2 end groups of PAMAM dendrimers or polyamide dendrons are attached to long chain hydrocarbons (i.e., lipids) at close to 100% conversion via amide or carbamate bonds, as in the above literature examples, the resulting amphiphilic dendritic materials are generally highly crystalline, and hence difficult to process into coherent thin films on substrates.